Stages Of Spermatogenesis Research Articles

Studi Literatur : Pengaruh Suhu terhadap Ikan Nila (Oreochromis niloticus)

Tilapia is one example of fish that is successfully cultivated in the world. Tilapia can live in deep and wide waters as well as in narrow and shallow ponds, and can live in rivers. The main environmental and management factors that affect the survival and growth of fish are dissolved oxygen levels, temperature, pH, feeding rate, and others. This study aims to determine the effect of temperature on tilapia (Orechromis niloticus). The method used in this research is a literature study (systematic review) by analyzing several scientific articles that are relevant to the study being discussed. The results of this study are known that temperature is very influential on several aspects of life, namely the level of survival, appetite, level of stability of the body for disease, growth rate, fish movement, viscosity and blood flow, oxygen consumption, leukocyte count, glucose level. in the blood, the number of erythrocytes, the number of platelets, the reaction of changing ammonium to ammonia, seed maintenance, tilapia weight, spermatogenesis stage in male individuals, hemoglobin concentration, feminization and masculinization of gonads, gastric evacuation rate, number of egg production, swimming speed, metabolic rate, response to feeding, the level of infertility in female fish, tilapia seed weight, fish stress level, the mechanism for determining the number of sex ratios, ovarian structure, and testicular structure. The conclusion of this study is that temperature greatly influences the life of tilapia (Oreochromis niloticus).

A novel pipeline for computerized mouse spermatogenesis staging.

Differentiating 12 stages of the mouse seminiferous epithelial cycle is vital towards understanding the dynamic spermatogenesis process. However, it is challenging since two adjacent spermatogenic stages are morphologically similar. Distinguishing Stages I-III from Stages IV-V is important for histologists to understand sperm development in wildtype mice and spermatogenic defects in infertile mice. To achieve this, we propose a novel pipeline for computerized spermatogenesis staging (CSS). The CSS pipeline comprises four parts: (i) A seminiferous tubule segmentation model is developed to extract every single tubule; (ii) A multi-scale learning (MSL) model is developed to integrate local and global information of a seminiferous tubule to distinguish Stages I-V from Stages VI-XII; (iii) a multi-task learning (MTL) model is developed to segment the multiple testicular cells for Stages I-V without an exhaustive requirement for manual annotation; (iv) A set of 204D image-derived features is developed to discriminate Stages I-III from Stages IV-V by capturing cell-level and image-level representation. Experimental results suggest that the proposed MSL and MTL models outperform classic single-scale and single-task models when manual annotation is limited. In addition, the proposed image-derived features are discriminative between Stages I-III and Stages IV-V. In conclusion, the CSS pipeline can not only provide histologists with a solution to facilitate quantitative analysis for spermatogenesis stage identification but also help them to uncover novel computerized image-derived biomarkers. https://github.com/jydada/CSS. Supplementary data are available at Bioinformatics online.

Different Strategies to Attenuate the Toxic Effects of Zinc Oxide Nanoparticles on Spermatogonia Cells.

Zinc oxide nanoparticles (ZnO NPs) are one of the most used nanoparticles due to their unique physicochemical and biological properties. There is, however, a growing concern about their negative impact on male reproductive health. Therefore, in the present study, two different strategies were used to evaluate the recovery ability of spermatogonia cells from the first stage of spermatogenesis (GC-1 spg cell line) after being exposed to a cytotoxic concentration of ZnO NPs (20 µg/mL) for two different short time periods, 6 and 12 h. The first strategy was to let the GC-1 cells recover after ZnO NPs exposure in a ZnO NPs-free medium for 4 days. At this phase, cell viability assays were performed to evaluate whether this period was long enough to allow for cell recovery. Exposure to ZnO NPs for 6 h and 12 h induced a decrease in viability of 25% and 41%, respectively. However, the recovery period allowed for an increase in cell viability from 16% to 25% to values as high as 91% and 84%. These results strongly suggest that GC-1 cells recover, but not completely, given that the cell viability does not reach 100%. Additionally, the impact of a synthetic chalcone (E)-3-(2,6-dichlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one (1) to counteract the reproductive toxicity of ZnO NPs was investigated. Different concentrations of chalcone 1 (0–12.5 µM) were used before and during exposure of GC-1 cells to ZnO NPs to mitigate the damage induced by NPs. The protective ability of this compound was evaluated through viability assays, levels of DNA damage, and cytoskeleton dynamics (evaluating the acetylated α-tubulin and β-actin protein levels). The results indicated that the tested concentrations of chalcone 1 can attenuate the genotoxicity induced by ZnO NPs for shorter exposure periods (6 h). Chalcone 1 supplementation also increased cell viability and stabilized the microtubules. However, the antioxidant potential of this compound remains to be elucidated. In conclusion, this work addressed the main cytotoxic effects of ZnO NPs on a spermatogonia cell line and analyzed two different strategies to mitigate this damage, which represent a significant contribution to the field of male fertility.

A novel testis-enriched gene, Samd4a, regulates spermatogenesis as a spermatid-specific factor.

Spermatogenesis is the highly orchestrated process involving expression of a series of testicular genes. Testis-enriched genes are critical for cellular processes during spermatogenesis whose disruption leads to impaired spermatogenesis and male infertility. Nevertheless, among poorly investigated testicular genes are the mouse Samd4a and human SAMD4A which were identified in the current study as novel testis-enriched genes through transcriptomic analyses. In particular, as orthologous alternative splicing isoforms, mouse Samd4a E-form and human SAMD4AC-form containing the SAM domain were specific to testes. Western blot analyses revealed that the murine SAMD4AE-form was predominantly found in the testis. Analyses on GEO2R and single-cell RNA-seq datasets revealed that the Samd4a/SAMD4A expression was enriched in spermatids among various types of cells in adult testes. To investigate in vivo functions of Samd4a, Samd4a knockout mice were generated using the CRISPR/Cas9 system. The Samd4a deficiency resulted in lower testis weight, absence of elongated spermatids, and an increased number of apoptotic cells. Profiling of gene expression in human testis samples revealed that the SAMD4A expression was comparable between obstructive azoospermia patients and normal controls, but significantly lowered in nonobstructive azoospermia (NOA) patients. Among three subgroups of NOA, pre-meiotic arrest (NOA-pre), meiotic arrest (NOA-mei), and post-meiotic arrest (NOA-post), expression level of SAMD4A was higher in the NOA-post than the NOA-mei, but there was no difference between the NOA-pre and NOA-mei. The current studies demonstrated spermatid stage-specific expression of Samd4a/SAMD4A, and impairment of the late stages of spermatogenesis by disruption of the mouse Samd4a gene. These data suggest that Samd4a/SAMD4A plays an essential role in normal spermatogenesis, and SAMD4A, as a spermatid specific marker, can be used for subcategorizing NOA patients. Further understanding the molecular role of SAMD4A will advance our knowledge on genetic regulations in male infertility.

Gap junctions mediate discrete regulatory steps during fly spermatogenesis.

Gametogenesis requires coordinated signaling between germ cells and somatic cells. We previously showed that Gap junction (GJ)-mediated soma-germline communication is essential for fly spermatogenesis. Specifically, the GJ protein Innexin4/Zero population growth (Zpg) is necessary for somatic and germline stem cell maintenance and differentiation. It remains unknown how GJ-mediated signals regulate spermatogenesis or whether the function of these signals is restricted to the earliest stages of spermatogenesis. Here we carried out comprehensive structure/function analysis of Zpg using insights obtained from the protein structure of innexins to design mutations aimed at selectively perturbing different regulatory regions as well as the channel pore of Zpg. We identify the roles of various regulatory sites in Zpg in the assembly and maintenance of GJs at the plasma membrane. Moreover, mutations designed to selectively disrupt, based on size and charge, the passage of cargos through the Zpg channel pore, blocked different stages of spermatogenesis. Mutations were identified that progressed through early germline and soma development, but exhibited defects in entry to meiosis or sperm individualisation, resulting in reduced fertility or sterility. Our work shows that specific signals that pass through GJs regulate the transition between different stages of gametogenesis.

Towards the mechanism of spermatotoxicity of p-tert-butyl-alpha-methylhydrocinnamic aldehyde: inhibition of late stage ex-vivo spermatogenesis in rat seminiferous tubule cultures by para-tert-butyl- benzoic acid.

Molecules metabolized to para-tert-butyl-benzoic acid (p-TBBA) affect male reproduction in rats through effects on spermatogenesis. This toxicity is specific to p-TBBA and not observed in meta-substituted analogues. The underlying mode of action was evaluated by comparing effects of p-TBBA and the position isomer m-TBBA (2-50µM) in an ex vivo 3D primary seminiferous tubule cell culture system from juvenile Sprague Dawley rats (Bio-AlteR®). Treated cultures were evaluated for CoA-conjugate formation, cytotoxicity, blood-testis barrier functionality and different germ cell populations to assess effects on spermatogenesis. In addition, an evaluation of the metabolome of treated cultures was performed by using MxP® Broad Profiling via a LC-MS/MS and GC-MS platform. Para-TBBA decreased germ cell populations of late stages of spermatogenesis and led to the formation of CoA-conjugates in the ex vivo tissue. In addition, p-TBBA had a pronounced effect on the metabolome by affecting lipid balance and other CoA-dependent pathways contributing to energy production and the redox system. Meta-TBBA did not affect germ cell populations and no m-TBBA related CoA-conjugates were detectable. The metabolic profile of m-TBBA treated cells was comparable to vehicle control treated cultures, indicating that formation of CoA-conjugates, inhibition of spermatogenesis, and effects on the metabolome are mechanistically linked events. Thus, for this specific chemical group an adverse outcome pathway can be postulated, including the formation of benzoic acid metabolites, accumulation of CoA-conjugates to a certain threshold and CoA depletion, which affects the metabolic and lipid profile and leads to tissue specific effects with impaired functionalities such as spermatogenesis.

The germ cell-specific RNA binding protein RBM46 is essential for spermatogonial differentiation in mice.

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for ‘RNA binding protein 46’ (RBM46). A highly evolutionarily conserved gene, Rbm46 is also essential for fertility in both flies and fish. We found Rbm46 expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generated Rbm46 knockout (KO, Rbm46-/-) mice; although male Rbm46-/- mice were viable and appeared grossly normal, they were infertile. Testes from adult Rbm46-/- mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and ‘enhanced crosslinking immunoprecipitation’ coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.

Développement Testiculaire chez Poisson Combattant (Betta splendens), variété de Demi-lune, Subis 35 jours de Différents Régimes de Photopériodes

Fighting fish (Betta splendens) is one of the fresh water ornamental fish with highly commercial value. This fish has a distinctive morphological shape which is aesthetic and is easy to maintain. Demand for this fish increased leading to opportunities for cultivation. Quality and quantity of fries were constantly needed, and this could be achieved by manipulating reproduction. Photoperiod regimes could play a role to do this. The purpose of this study was to determine the effect of photoperiods on the testis development of halfmoon strains of fighting fish.
 A completely randomized design was run to test 3 treatments in quintuplicates. The fish were reared under treatment P1 (10 hours light L and 14 hours dark D), treatment P2 (14 hours L and 10 hours D) and treatment P0 (12 hours L and 12 hours D). The fish were maintained for 35-d, in Aquatic Resources Laboratory of FPIK Unsoed. The results showed that treatment P2 was significantly different (P<0.05), which the highest spermatogenesis stage (54.78 ± 16.64%) was obtained. The study concluded that male fighting fish under P2 photoperiod could accelerate testicular maturation.

Effect of isotretinoin (Netlook) on the testis of adult male albino rats and the role of omega 3 supplementation: Ahistological and biochemical study.

Isotretinoin is an oral retinoid which used across the world in the treatment of patients especially adolescents complaining of acne. In spite of the prevalent clinical use of isotretinoin, the generation of oxidative stress with the affection of several organs leads to the limitation and restriction of its use. Omega‐3 (N‐3) is an essential polyunsaturated fatty acid (PUFAs) with powerful antioxidant properties. The aim of this study was to investigate the histological and biochemical changes occurring in the rat testis following isotretinoin intake and to evaluate the role of omega 3 supplementation in ameliorating testicular damage. Thirty adult male albino rats were divided equally into three groups. Group I is the control group, group II received isotretinoin (1.0 mg/kg/day) dissolved in distilled water and group III received isotretinoin (1.0 mg/kg/day) and omega 3 (400 mg/kg/day). Testis samples were collected and processed for light and electron microscopic examination. The blood samples were collected for biochemical assessments. Results indicated that isotretinoin caused histological changes in all stages of spermatogenesis and alterations of the hormonal assay. These changes in the rat testis which were corrected by omega 3 use.

Histological characteristics of spermatogenesis stages in annual reproductive cycles of male Siberian sturgeons in various types of industrial fish farming

The modern development of aquaculture industry is inseparably associated with brood stock formation and reproduction, breeding of seeding and commercial production. Specific breeding conditions, with longer periods of water temperatures favourable for fish growth, simultaneously affect the development of the reproductive system of fish. Studies of these processes enable the best approach to obtaining reproductive products whenever the farm needs them. The study was performed at the Konakovo Division of the Branch for Freshwater Fisheries of the Federal State Budget Scientific Institution ‘Russian Federal Research Institute of Fisheries and Oceanography’ (VNIIPRKH), in a recirculating aquaculture system (RAS) and flowthrough industrial farm. Even-aged Siberian sturgeons of generation 2017 were used in the study. Samples were taken by biopsy and fixed in 70% ethanol. The paper presents the data on spermatogenesis in Siberian sturgeons in a flow-through industrial farm with annual thermal constant of 5600 degree days and with combination housing of brood fish. During the first stage, the fish are kept in a recirculating aquaculture system for 210 days, with the thermal constant of 1511 degree days, with subsequent housing and breeding in a flow-through farm for 150 days, with the thermal constant of 2340 degree days. The histological characteristics of spermatogenesis and reproductive cycles of Siberian sturgeons are presented, with a detailed description and illustrations of the processes and respective differences depending on housing conditions. The studies showed that specific temperature conditions can prolong the spawning season by 2–3 months by shifting the reproductive cycles of male Siberian sturgeons at first spawning with combination housing in flow-through tanks and RAS (prolonged wintering conditions).

Seasonal changes in the expression of molecular markers of stallion germ cells

The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understand the process of sperm production. Furthermore, comparing internal and external factors related to spermatogenesis during the breeding season (BS) and NBS may provide a solution for subfertile/infertile stallions. It is especially pertinent to study the expression pattern of different protein markers during undifferentiated, differentiating, and differentiated spermatogonia. Deleted in azoospermia-like (DAZL), undifferentiated cell transcription factor 1 (UTF-1), and protein gene product 9.5 (PGP9.5) are the molecular markers expressed at different stages of spermatogenesis. However, whether the expression pattern of these molecular markers is similar throughout the year in stallion remains undetermined. The objectives of this study were to (1) investigate the expression pattern and localization of DAZL, UTF-1, and PGP9.5 within seminiferous tubules and (2) evaluate the relative mRNA levels of these three germ cell markers in stallion testes during BS and NBS. Immunohistochemistry was performed to check and compare the expression pattern and localization of DAZL, UTF-1, and PGP9.5 antibodies. Reverse transcription-quantitative PCR analysis was performed to calculate the relative mRNA expression levels in the testes. Testicular tissues from thoroughbred stallions were collected during routine castration that was carried out in field conditions. Immunostaining of germ cells with DAZL and UTF-1 in BS and NBS were not significantly different. However, the relative mRNA expression levels of DAZL and UTF-1 were significantly different in both groups. Interestingly, the immunolabeling and the relative mRNA expression of PGP9.5 were significantly different between BS and NBS. From these results, it is hypothesized that the expression level of these putative molecular markers might be gonadotropin-dependent in stallion testes.

Impact of substerilizing dose on histological changes in gonads and ovaries of Ephestia cautella (Lepidoptera: Pyralidae) by gamma radiation

To investigate the effect of Gamma radiation on the reproductive systems of emerged female and male of Ephestia cautella (Walker) moths, mature pupae of both sexes were irradiated with doses of 50, 100 and 150 Gy. Histological study of the treated individuals showed in females that the ovaries appear sever damage in the follicular epithelium at all doses, which become thinness and separated from developing oocytes, moreover, some of the nurse cells were rupture. In males which treated with 150 Gy, it was noticed retardation in the stages of spermatogenesis and few numbers of sperm bundles and their dispersion in the testicular follicles’ adults, on the other hand, the doses of 50 and 100 Gy showed little or moderate effects on the structure of the testis contents.

A systematic review of retinoic acid in the journey of spermatogonium to spermatozoa: From basic to clinical application

Background: Retinoic acid plays an essential role in testicular development and functions, especially spermatogenesis. We have reviewed the role of retinoic acid from basic (molecular) to clinical application. Methods: A search was conducted in the online database including PubMed, Google Scholar, and Scopus for English studies published in the last eight years about this issue. We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in assessing the studies we are going to investigate. Results: Studies indicated that retinoic acid plays an essential role during pluripotent stem cell migration and lineage commitment, cell differentiation, apoptosis, stem cell number regulation, and maturation arrest in spermatogenic cells. Retinoic acid can also affect related protein expression and signaling pathways at different stages of spermatogenesis. Four studies have applied retinoic acid to humans, all of them in the single-arm observational study. The results look promising but need further research with more controlled study methods, randomization, and large samples. Conclusions: This current systematic review emphasizes a novel retinoic acid mechanism that has not been well described in the literature previously on its functions during the first seven days of spermatogenesis, leading to new directions or explanations of male infertility cause and treatments as a part of reproductive health care.

Proteome Informatics in Tibetan Sheep (Ovis aries) Testes Suggest the Crucial Proteins Related to Development and Functionality.

Testis has an indispensable function in male reproduction of domestic animals. Tibetan sheep (Ovis aries) is a locally adapted breed of sheep raised in the Qinghai-Tibet Plateau, with outsized roles in providing the livelihood for millions of residents. Nevertheless, less is known on how protein expression and their functional roles in developmental testes of such breed limit their use in breeding efforts. In this study, we obtained comprehensive protein profiles from testes of Tibetan sheep at three developmental stages (including pre-puberty, post-puberty, and adulthood) using data-independent acquisition-based proteomic strategy to quantitatively identify the differentially abundant proteins (DAPs) associated with testicular development and function and to unravel the molecular basis of spermatogenesis. A total of 6,221 proteins were differentially expressed in an age-dependent manner. The reliability of the gene expression abundance was corroborated by quantitative PCR and targeted parallel reaction monitoring. These DAPs were significantly enriched to biological processes concerning spermatid development and sperm deformation, mitosis, glycolytic process, cell-cell/extracellular matrix (ECM) junctions, cell proliferation, apoptosis, and migration and to the pathways including, developmental process and sexual reproduction-related (such as VEGF, estrogen, insulin, GnRH, Hippo, PI3K-Akt, mTOR, MAPK, and AMPK), and testicular cell events-related pathways (such as tight/gap/adherens junctions, ECM-receptor interaction, regulation of actin cytoskeleton, glycolysis, cell cycle, and meiosis). Based on these bioinformatics analysis, we constructed four protein–protein interaction network, among which the proteins are involved in mitosis, meiosis, spermiogenesis, and testicular microenvironment, respectively. Altogether, these bioinformatics-based sequencing results suggest that many protein-coding genes were expressed in a development-dependent manner in Tibetan sheep testes to contribute to the testicular cell development and their surrounding microenvironment remodeling at various stages of spermatogenesis. These findings have important implications for further understanding of the mechanisms underlying spermatogenesis in sheep and even other plateau-adapted animals.

Reproductive and developmental toxicity screening test of new TiO2 GST in Sprague-Dawley rats

TiO2 nanoparticles are widely used in paints, plastics, cosmetics, printing ink, rubber, food products, pharmaceuticals and other products (photocatalyst, etc.). However, there is little toxicological information during reproduction and developmental period. This study was performed to obtain safety data for new TiO2 powder, GST (Green Sludge Titanium) produced through sludge recycling of the sewage treatment plant for Reproduction/developmental toxicity screening test in Sprague-Dawley (SD) rats in according to the OECD test guideline (TG 421). Based on the results of the dose-range finding study (14-day repeated toxicity), GST was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg B.W/day. Males were dosed for 35 days beginning 14 days before mating, and females for a maximum of 53 days beginning 14 days before mating to day 13 of lactation, including throughout the mating, gestation and lactation periods. In the reproductive and developmental examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic / microscopic findings, stages of spermatogenesis in the testis, reproductive finding (estrous cycle, copulation-fertility-gestation index), developmental finding (number of corpora lutea and implantations, pups parameters including live birth and viability index). The NOAEL for reproductive/developmental screening toxicity was concluded to be 2000 mg/kg/day under the present study conditions.

P-081 Effect of chemotherapy (cytarabine) and acute myeloid leukemia on the development of spermatogenesis, spermatogonial microenvironment and inflammatory factors at the adult age of immature-treated mice

Abstract Study question How do AML and cytarabine affect the development of spermatogenesis, germ cell microenvironment and testicular inflammatory factors at the adult age of immature-treated mice? Summary answer AML and cytarabine significantly damaged the normal histology of the seminiferous tubules and impaired different stages of spermatogenesis, testicular growth/inflammatory factor in the adult age. What is known already The effect of different types of cancers on male fertility was reported. AML constitutes around 20% of the diagnosed childhood leukemia. Cytarabine is used as anti-AML treatment. Cytarabine is involved in reduction of testes growth and induces testicular atrophy in adults. In adult AML patients, it was shown that AML affects semen parameters even before chemotherapy treatment. Furthermore, a significant reduction in semen quality of thawed cryopreserved semen from leukemia patients compared to control patients was reported. However, the effect of AML and cytarabine before puberty on the development of spermatogenesis in adult age was not yet reported. Study design, size, duration Two-week-old males C57/BLACK mice were used. 1. Control group – injected with saline. 2. AML group – injected (intraperitoneal; i.p) with 3x104 AML cells/100 ml (murine C-1498 AML cell line). 3. Cytarabine group - three injections of cytarabine (140 mg/kg/100 ml) were performed every 12 hours. 4. AML+cytarabine group – cytarabine was i.p injected 24 hours after AML injection (injections were performed every 12 hours 3 times). Mice were sacrificed 1,2,3,4,5 weeks after AML injection. Participants/materials, setting, methods Testes were removed, weighted and fixed in Bouin’s solution for histological evaluation, or kept at -70°C for RNA extraction. The presence of premeiotic (SALL4, PLZF), meiotic (CREM-1) and postmeiotic (ACROSIN) cells were examined by immunofluorescence staining analysis and/or RNA expression by qPCR analysis. Main results and the role of chance We have successfully developed a system of AML in immature mice by i.p injection of C1498 cells. Cytarabine prolonged life of AML-treated mice from 2.5 weeks to 4 weeks. AML had no effect on both body and testicular weight. However, cytarabine significantly reduced testicular weight (around 50% of the control in the first 3 weeks after treatment, and around 30% of the control from 3-4.5 weeks after treatment; p < 0.001). One and/or 2 weeks after the treatment showed that both AML and cytarabine alone or in combination significantly decreased seminiferous tubules normal histology compared to control, (30%, 20%, 10% respectively) (p < 0.001). Cytarabine, but not AML significantly increased the expression of PLZF and SALL4 after 2 weeks compared to control (p < 0.001). However, each treatment significantly increased the percentages of seminiferous tubules with apoptotic cells compared to control (p < 0.05). AML or cytarabine significantly decreased the percentages of tubules with meiotic and postmeiotic markers compared to control (p < 0.05). Also, AML or cytarabine significantly decreased the testicular protein levels of GDNF, CSF-1 (p < 0.01) but did not affect LIF compared to control. Furthermore, AML or cytarabine significantly decreased the protein levels of IL-10. However, AML significantly increased IL-6 when cytarabine increased it compared to control (p < 0.01). Limitations, reasons for caution Animal model of leukemia may behave different from human model. Wider implications of the findings We successfully developed AML disease in immature mice, and demonstrated its impairment the normal histology of the seminiferous tubules and spermatogenesis in the adult age. We showed that AML and cytarabine affect testicular microenvironmental and inflammatory factors. Our results may assist in development future therapeutic strategies for male fertility preservation. Trial registration number NON

O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p < 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p < 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable

Novel copy number variations within SYCE1 caused meiotic arrest and non-obstructive azoospermia

BackgroundNon-obstructive azoospermia (NOA) is the most severe disease in male infertility, but the genetic causes for majority of NOA remain unknown.MethodsTwo Chinese NOA-affected patients were recruited to identify the genetic causal factor of infertility. Whole-exome sequencing (WES) was conducted in the two patients with NOA. Sanger sequencing and CNV array were used to ascertain the WES results. Hematoxylin and eosin (H&E) staining and immunofluorescence (IF) were carried out to evaluate the stage of spermatogenesis arrested in the affected cases.ResultsNovel heterozygous deletion (LOH) within SYCE1 (seq[GRCh37] del(10)(10q26.3)chr10:g.135111754_135427143del) and heterozygous loss of function (LoF) variant in SYCE1 (NM_001143763: c.689_690 del:p.F230fs) were identified in one NOA-affected patient. While homozygous deletion within SYCE1 (seq[GRCh37] del(10)(10q26.3)chr10:g.135340247_135379115del) was detected in the other patient with meiotic arrest. H&E and IF staining demonstrated that the spermatogenesis was arrested at pachytene stage in the two patients with NOA, suggesting these two novel CNVs within SYCE1 could lead to meiotic defect and NOA.ConclusionsWe identified that two novel CNVs within SYCE1 are associated with meiotic arrest and male infertility. Thus, our study expands the knowledge of variants in SYCE1 and provides a new insight to understand the genetic etiologies of NOA.

Spatiotemporal Regulation of a Single Adaptively Evolving Trans-Regulatory Element Contributes to Spermatogenetic Expression Divergence in Drosophila.

Due to extensive pleiotropy, trans-acting elements are often thought to be evolutionarily constrained. While the impact of trans-acting elements on gene expression evolution has been extensively studied, relatively little is understood about the contribution of a single trans regulator to interspecific expression and phenotypic divergence. Here, we disentangle the effects of genomic context and miR-983, an adaptively evolving young microRNA, on expression divergence between Drosophila melanogaster and D. simulans. We show miR-983 effects promote interspecific expression divergence in testis despite its antagonism with the often-predominant context effects. Single-cyst RNA-seq reveals that distinct sets of genes gain and lose miR-983 influence under disruptive or diversifying selection at different stages of spermatogenesis, potentially helping minimize antagonistic pleiotropy. At the round spermatid stage, the effects of miR-983 are weak and distributed, coincident with the transcriptome undergoing drastic expression changes. Knocking out miR-983 causes reduced sperm length with increased within-individual variation in D. melanogaster but not in D. simulans, and the D. melanogaster knockout also exhibits compromised sperm defense ability. Our results provide empirical evidence for the resolution of antagonistic pleiotropy and also have broad implications for the function and evolution of new trans regulators.

Effect of Palm Pollen and Pomegranate Peel Supplementation on Sperm Development in Male Rabbits.

The use of plants, plant extracts, and folk remedies for the treatment of human diseases has been known as traditional medicine. It is approved that the use of herbal medicine dates far back to 60,000 years ago in Iraq. This study aimed to investigate the possible effects of pomegranate and palm pollen, as two powerful herbal remedies with antioxidant, anti-inflammatory, and anti-bacterial effects, on fertility in the male rabbits. A total of 15 healthy New Zealand White male adult rabbits were randomly assigned to three groups (n=5 in each group). Group A: 5 rabbits did not receive herbal treatment (control group). Group B: 5 rabbits were treated with palm pollen at a dose of 2 g by oral gavage daily. Group C: 5 rabbits were treated with pomegranate peel (2 g) treated by oral gavage daily. All animals were euthanized 90 days after the beginning of the experiment. Following the euthanasia, the testis was removed. For sperm recovery, the epididymis was dissected from the testicles and the tail of the epididymis was minced in the sperm washing medium and incubated for 15 min in an atmosphere of humidified 5% CO2, 95% The samples were evaluated by using computerized semen analyzer apparatus (CASA). After 90 days, the treated groups with palm pollen and pomegranate peel (groups B and C) demonstrated a significant increase in the sperm concentration, compared to group (A). Moreover, the recorded data revealed that the sperm concentration in group C was significantly higher than that in group B. The study indicated that the total sperm motility was increased significantly in groups B and C, as compared to that in the control group, while the total sperm motility in group C increased significantly, compared to group B. The histological evolutions signified that in the control group, the epithelial of seminiferous tubules were at different stages of spermatogenesis, the spermatocytogenesis (primary and secondary) with the spermogenesis (spermatids) were found in a cluster at the cavity of the tubule. Furthermore, the results of histological evaluations in groups B and C were distinguished by a significant augmentation of the seminiferous tubules with primary and secondary spermatocytes, as well as some degrees of spermogenesis (round spermatids), compared to group A. Moreover, increased angiogenesis was observed in groups B and C. In conclusion, the results pointed out that herbal remedies have a significant effects in the treatment of reproductive disorders.